The characteristics of light emanating from an object or a material may be advantageously detected and analyzed in order to determine characteristics of the object or material under examination. For many years, spectrographic techniques have been used to perform analysis of materials ranging from human blood and other biological materials to slag from a crucible. For example, it has been known that wavelengths of light absorbed by a material, as well as the wavelengths of light emitted by a material during an excited state, such as combustion, both indicate the composition of the material. Today, analytic instruments in industrial, scientific and medical applications make widespread use of such emission spectra and absorption spectra. Another such technique is Raman spectroscopy, where, for example, the output of a mercury vapor arc lamp may be filtered and used to excite a transparent material. As the light transmits through the material, it is scattered and undergoes a change in wavelength and a random alteration in phase due to changes in the rotational or vibrational energy of the sample. Raman scattering is a principal analytic tool in industry and science today.
A specific class of analytic instruments uses fluorescence (or phosphorescence) to identify materials. In such systems, an excitation source, such as a laser, is used to excite atoms or molecules, raising electrons into higher energy states. When the electrons revert back to the unexcited state, they fluoresce or emit photons of light characteristic of the excited atom or molecule. In addition, the time delay between the exciting light and the emitted light, as well as the amplitude of the emitted light, provide information about the material's composition, lifetimes, and concentration of various components. Instruments that provide this function are known as frequency domain fluorometers or time correlated single photon counting instruments (TCSPC).
Frequency domain fluorometers rely on phase delay and amplitude measurements. The exaltation source is modulation which causes the re-emission of a fluorescent signal and it is the relationship between the re-emission (phase delay) and reduction in modulation which is used to calculate the lifetime. In one class of instruments such measurements may be achieved by frequency modulating a light source. For example, one may employ for this purpose a pulsed dye laser, or a continuous wave laser whose output is externally modulated by a Pockels cell or an LED which is intrinsically modulated.
By “phase” is meant the re-emission delay in degrees or time, of the modulated fluorescence emission of an unknown sample as compared to a modulated reference, which may be either the excitation source or a known sample. By “modulation”, sometimes also referred to as the modulation ratio, is meant the ratio of the amplitude of a fixed reference, either a known sample or the excitation source, to the fluorescence amplitude of the unknown sample. A further refinement of the measurement technique is to perform the measurement of modulation and phase on a sample many times using different modulation frequencies each time. Generally, this results in the generation of a first characteristic for phase as a function of modulation frequency and a second characteristic of modulation as a function of modulation frequency. Generally, phase angle will increase with increasing modulation frequency. Moreover, for samples exhibiting longer lifetimes, phase will be larger at a given modulation. Similarly, modulation tends to decrease for samples exhibiting longer lifetimes, at a given modulation frequency.
If curve fitting techniques are used to match the plot of frequency versus phase and the plot of frequency versus modulation, to a pair of equations, analysis of the equations can be used to discern multiple individual fluorescing components, for example organic molecules, fluorescing semiconductor depositions or dopants or the like, in a sample. Curve fitting techniques are known in the field today and generally involve the use of a digital computer to perform the desired curve fitting and the comparison of various physical models that represent the molecular system and its environment.
Frequency domain cross-correlation techniques are well developed in the prior art (e.g. U.S. Pat. Nos. 4,840,485, 5,151,869 and 5,196,709 etc.) and commercial instruments are available for sale. The downside of these techniques are that each frequency is individually scanned, and this is a slow process. Additionally only one detector at a time is used, and one loses any spatial relationships within the samples.
To maintain the spatial relationships with the sample another technique has been developed called fluorescence lifetime imaging microscope (FLIM), using a single frequency domain instrument coupled to a camera using homodyne and sometimes heterodyne detection.
A further improvement on these technique was disclosed by Mitchell in U.S. Pat. No. 4,937,457. Mitchell disclosed a technique of producing multiple coherent harmonics to speed up the process of data collection. In these instruments, fluorescence measurement is obtained by deriving phase and modulation information in the steady state from a fluorescence or phosphorescence emission driven by a light source modulated with multiple modulation frequencies.
In another class of instruments, which rely on time-domain lifetime measurement, a time correlated single photon counting (TCSPC) method is employed. In this type of instrument, a measurement is made of the probability of a fluorescent photon emission after the fluorophore receives an excitation pulse. The measurement is made by counting the arrival time of individual photons within certain time periods after emission.
The light sources for both of these instruments suffer from similar drawbacks; are expensive and their light sources can be large and require special facilities and operator training and so forth.
In accordance with the invention a method of spectrographic measurement comprises generating light energy using a solid state low capacitance excitation source. The light energy is caused to fall on a sample to be assayed, causing the sample to output an output optical signal. A plurality of modulation frequencies are generated. In addition, a plurality of heterodyne frequencies are generated to form a set of heterodyne signals at heterodyne frequencies. Each of the heterodyne frequencies is associated with one of the modulation frequencies. The modulation frequencies are coupled to the excitation source, causing the excitation source to generate excitation energy modulated in intensity in proportion to the modulation frequencies. A portion of the substantially incoherent excitation energy is sampled to form a substantially incoherent reference excitation signal. The output optical signal is focused as an image modulated with the plurality of modulation frequencies on an image intensifier. The image is intensified to form an intensified image modulated with the plurality of modulation frequencies. The intensified image modulated with the plurality of modulation frequencies is received on a multi-element optical detector. A plurality of measurement signals are generated using the multi-element optical detector. Each measurement signal is associated with one of the elements. Each measurement signal associated with one of the elements of the multi-element optical detector is mixed with the heterodyne signal to generate a plurality of low-frequency measurement modulation products. A low-frequency measurement modulation product is associated with each of the modulation frequencies and comprises the difference between a single modulation frequency and its associated heterodyne frequency and has a measurement amplitude and phase. The substantially incoherent reference excitation energy is mixed with the heterodyne signal to generate a plurality of reference modulation products, one reference modulation product being associated with each of the modulation frequencies and comprising the difference between a single modulation frequency and its associated heterodyne frequency and having a reference amplitude and phase, each of the low-frequency reference modulation products being associated with one of the measurement modulation products. Each of the plurality of low-frequency measurement modulation products is compared to its associated low-frequency reference modulation product to generate an output signal indicating characteristics of the sample at the region on the sample associated with each of the elements.